Project Summary/Abstract The proposed research focuses on how H. pylori, along with gastric acidity, is able to injure the gastric mucosa and trigger development of advanced disease. H. pylori infection is highly prevalent worldwide and at a minimum causes gastric inflammation. Some of those infected will progress to develop gastric or duodenal ulcer disease, gastric atrophy, and cancer. Treatment is becoming more difficult with emerging antibiotic resistance. It is not definitively known how the bacteria are able to evade the immune system, leading to lifelong infection, or what factors contribute to development of advanced disease, although multiple bacterial and host factors have been studied. The objective of this research is to develop a new model system for co- culture of H. pylori with gastric cells, then to use this model to study the effect of H. pylori on a specific cell adhesion protein, the Na,K-ATPase and to study the movement of H. pylori lipopolysaccharide (LPS) through the epithelial barrier. Preliminary findings suggest that the presence of H. pylori leads to downregulation of the Na,K-ATPase, which would lead to weakening of connections between cells. H. pylori appears to specifically interfere with interactions between the transporter subunits and endoplasmic reticulum chaperones. This result will be confirmed, mechanism investigated, and the effect on barrier function determined. The ?1 subunit of the Na,K-ATPase, involved with connections between cells, and the ?1 subunit, which connects with the cytoskeleton, will be overexpressed in gastric epithelial cells to determine if changes in barrier function indcued by H. pylori can be overcome. The possibility that post-translational modifications to the Na,K-ATPase or associated proteins are involved in the mechanism of downregulation will be investigated by specialized quantitative mass spectometry techniques. LPS is a component of the outer membrane of gram negative bacteria, including H. pylori. The presence of LPS facilitates chronic infection and may interfere with barrier function. The movement of LPS through the epithelial barrier will be studied as a means of understanding gastric injury and possibly distal consequences of infection. The goal of this research is to better understand the mechanisms of gastric injury and advanced disease associated with H. pylori infection, which will lead to discovery of better treatment targets and gastric protective measures.